NSUN2-mediated m⁵C methylation of IRF3 mRNA negatively regulates type I interferon responses during various viral infections

5-Methylcytosine (m⁵C) is a widespread post-transcriptional RNA modification and is reported to be involved in manifold cellular responses and biological processes through regulating RNA metabolism. However, its regulatory role in antiviral innate immunity has not yet been elucidated. Here, we report that NSUN2, a typical m⁵C methyltransferase, negatively regulates type I interferon responses during various viral infections, including SARS-CoV-2. NSUN2 specifically mediates m⁵C methylation of <i>IRF3</i> mRNA and accelerates its degradation, resulting in low levels of IRF3 and downstream IFN-β production. Knockout or knockdown of NSUN2 enhanced type I interferon and downstream ISGs during various viral infection <i>in vitro</i>. And <i>in vivo</i>, the antiviral innate response is more dramatically enhanced in <i>Nsun2^+/−</i> mice than in <i>Nsun2^+/+</i> mice. The highly m⁵C methylated cytosines in <i>IRF3</i> mRNA were identified, and their mutation enhanced cellular <i>IRF3</i> mRNA levels. Moreover, infection with Sendai virus (SeV), vesicular stomatitis virus (VSV), herpes simplex virus 1 (HSV-1), or Zika virus (ZIKV) resulted in a reduction of endogenous NSUN2 levels. Especially, SARS-CoV-2 infection (WT strain and BA.1 omicron variant) also decreased endogenous levels of NSUN2 in COVID-19 patients and K18-hACE2 KI mice, further increasing type I interferon and downstream ISGs. Together, our findings reveal that NSUN2 serves as a negative regulator of interferon response by accelerating the fast turnover of <i>IRF3</i> mRNA, while endogenous NSUN2 levels decrease during SARS-CoV-2 and various viral infections to boost antiviral responses for effective elimination of viruses.5-甲基胞嘧啶（5-Methylcytosine，m⁵C）是一种广泛存在的转录后RNA修饰，据报道可通过调控RNA代谢参与多种细胞应答与生物学过程。然而，其在抗病毒天然免疫中的调控作用迄今尚未阐明。本研究发现，典型的m⁵C甲基转移酶NSUN2可在包括新型冠状病毒（SARS-CoV-2）在内的多种病毒感染过程中负调控I型干扰素应答。NSUN2特异性介导干扰素调节因子3（Interferon Regulatory Factor 3，IRF3）mRNA的m⁵C甲基化修饰，并加速其降解，最终导致IRF3及下游干扰素β（IFN-β）的表达量降低。体外实验中，敲除或敲低NSUN2可增强多种病毒感染下的I型干扰素应答及其下游干扰素刺激基因（Interferon-stimulated genes，ISGs）的表达。体内实验方面，Nsun2^+/-小鼠的抗病毒天然免疫应答较野生型Nsun2^+/+小鼠更为显著。研究人员鉴定出IRF3 mRNA上高度甲基化的m⁵C胞嘧啶位点，突变这些位点可提升细胞内IRF3 mRNA的水平。此外，仙台病毒（Sendai virus，SeV）、水疱性口炎病毒（vesicular stomatitis virus，VSV）、单纯疱疹病毒1型（herpes simplex virus 1，HSV-1）及寨卡病毒（Zika virus，ZIKV）感染均可降低内源性NSUN2的表达水平。尤为重要的是，新型冠状病毒野生株（WT strain）及BA.1奥密克戎变异株（BA.1 omicron variant）感染可降低新冠肺炎患者及K18-hACE2基因敲入（KI）小鼠体内的NSUN2水平，进一步增强I型干扰素应答及其下游干扰素刺激基因的表达。综上，本研究揭示NSUN2可通过加速IRF3 mRNA的快速降解作为I型干扰素应答的负调控因子，而在新型冠状病毒及多种病毒感染过程中，内源性NSUN2的表达水平会降低，从而放大抗病毒天然免疫应答以高效清除病毒。